JOURNAL OF VIROLOGY, Dec. 1991, p. 6461-6467

Vol. 65, No. 12

0022-538X/91/126461-07$02.00/0 Copyright © 1991, American Society for Microbiology

Broadly Neutralizing Antibodies Targeted to Mucin-Type Carbohydrate Epitopes of Human Immunodeficiency Virus JOHN-ERIK STIG HANSEN,'* CLAUS NIELSEN,2 MAIKEN ARENDRUP,1 SIGVARD LARS MATHIESEN,1 JENS OLE NIELSEN,' AND HENRIK CLAUSEN3

OLOFSSON,1

Department of Infectious Diseases 144, Hvidovre Hospital, DK-2650 Hvidovre,' and Department of Virology, State Serum Institute,2 and Department of Oral Diagnostics, The Royal Dental College,3 Copenhagen, Denmark Received 10 June 1991/Accepted 19 August 1991

The cancer-related mucin-type carbohydrate neoantigen Tn was found on gpl60 and gpl20 of human immunodeficiency virus (HIV). Immunoglobulin G (IgG) and IgM monoclonal antibodies (MAbs) against Tn neutralized infection with cell-free virus and blocked fusion between HIV-infected and uninfected cells. This inhibition was found in infection of both lymphocytic cells and monocytoid cells. Viruses tested included six HIV-1 and five HIV-2 isolates propagated in different cells, as well as infectious plasma from AIDS patients. The antiviral effect of anti-Tn MAbs occurred by specific binding of the MAb to the virus; this binding was inhibitable by pure Tn antigen, and indications were found that this inhibition occurred at a pre-entry step. Boosting the naturally occurring low-titer anti-Tn activity may be of prophylactic value, as suggested by the in vitro neutralization found in this study. Human immunodeficiency virus (HIV) is an enveloped virus containing two glycoproteins in its envelope. These two glycoproteins, gp120 and gp4l, are responsible for attachment and penetration of HIV, initiating infection of target cells, which are primarily CD4+ lymphocytes and monocytes-macrophages (10, 19). In natural infection, a humoral immune response is evoked and antibodies against both envelope glycoproteins are produced. Antibodies directed against an immunodominant region of gpl20 (amino acids 302 to 337) block infection (4, 16, 29). This region, however, is highly variable in its peptide composition, and neutralizing antibodies to the region have been found to be type specific (16, 26). Recently we have reported that monoclonal antibodies (MAbs) against some of the peripheral carbohydrate structures of the major envelope glycoprotein, gpl20, also block infection in vitro (12). Use of the viral carbohydrates, which are not structurally encoded by the viral genome, as targets for neutralizing antibodies could present a new possibility for group-specific vaccine development because this approach may be less sensitive to viral mutations and subsequent emergence of escape mutants and because carbohydrate neutralization epitopes may be expressed on a broader range of HIV isolates. Interest would naturally focus on carbohydrate structures which are specific for virus or infected cells. We have previously demonstrated the presence on gpl20 of an 0-linked carbohydrate antigen, sialosyl-Tn (NeuAc-GalNAc-Ser/Thr), and antibodies to this antigen neutralize infection of lymphocytes but enhance infection of U937 cells (12, 13). We therefore looked for the presence of the closely related carbohydrate antigen Tn (GalNAc-Ser/Thr), which is a known cancer-related neoantigen (17, 30) and examined whether MAbs against this epitope could neutralize or enhance infection in vitro with a variety of HIV isolates. The Tn antigen is of special interest not only because it is very restrictively expressed in healthy humans (17) but also because it can induce both humoral and *

cellular immune responses, which protect mice on challenge with Tn-expressing tumor cells (28). Additionally, the Tn antigen is relatively easy to produce synthetically, in contrast to sialosyl-Tn.

MATERIALS AND METHODS MAbs. Hybridoma supernatants (25 jig of immunoglobulin [Ig] per ml) containing MAbs 1E3 (IgG2a) and TKH6 (IgM) against Tn antigen (Sa) were used for initial experiments after extensive dialysis against phosphate-buffered saline (PBS) and filtration (pore size, 0.22 ,um). Results obtained with 1E3 hybridoma supernatants were subsequently confirmed by using MAb purified by protein A-Sepharose chromatography. Isotype-matched control MAbs for infectivity experiments were TH1 (6) and TKH5 (17a). Prior to use in infectivity assays, the toxicity of the MAbs in MT-4 cells was examined by trypan blue exclusion. None of the MAbs had any effect on the vitality or growth of the cell cultures, even at the highest concentration used in infectivity assays (Table 1). Cells. The CD4+ lymphocyte cell lines MT-4 (15), H9 (25), and HUT-78 (11) and the monocytoid cell line U937 (18, 32) were cultured at 37°C and 5% CO2, in RPMI 1640 with 10% heat-inactivated fetal calf serum (5% for U937), 2 ,uM glutamine, 100 IU of penicillin per ml, 20 ,ug of gentamicin per ml, and 100 IU of streptomycin per ml (growth medium). Cells were maintained at a concentration of 2 x 105 to 1 x 106 cells per ml, and the medium was exchanged twice weekly. Peripheral blood mononuclear cells from healthy donors were separated from blood by Ficoll-Hypaque centrifugation and stimulated for 3 days with phytohemagglutinin in growth medium containing 20 IU of interleukin-2 per ml. Virus. Supernatants from H9 cells infected with the HIV-1 HIVIIIB strain (25), from HUT-78 cells infected with HIV-2 SBL6669 (9), from 6D5 cells infected with HIV-1 CDC451 (8), or from MT-4 cells infected with HIV-2 LAV-2 (7) were filtered (pore size, 0.22 ,um), aliquoted, and stored at -80°C until use. The HIVIIIB isolate was also passed twice in

Corresponding author. 6461

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TABLE 1. Cell proliferation was unaffected by anti-carbohydrate MAbs in the highest concentration used in infectivity inhibition assays'

percent infection relative to control 120

-

Mean no. of live cells (104/ml) ± SEM in:

Day

20 23 118 182

0 1 4 7

2 2 14

20 24 142 172

100 -

PBS

1E3

TKH6

1 3 18 16

20 24 137 187

2 1 12 17

80 -

a One million MT-4 cells were incubated with PBS or 2 ,ug of MAb 1E3 or TKH6, and 200,000 cells in 1 ml were transferred in quadruplicate to a 24-well cell culture plate.

60 -

19

± ± ± ±

T

40 -

peripheral blood mononuclear cells from blood type A, B, or O donors as described previously (3), and supernatants (HIVIIIB/lyA, HIVIIIB/lyB, and HIVIIIB/lyO, respectively) from these cultures were stored as above. HIV-1 SSIOO2, which has previously been found to infect the monocytoid U937 cell line readily (14), and three fresh HIV-1 isolates, SSI121c, SSI122c, and SSI146c, were passed in MT-4 cells, whereas three fresh HIV-2 isolates, SS1082, SS1131 and SS1502, were passed in CD4+ donor lymphoblasts. Before use, the 50% cell culture infective dose (CCID50) of the virus preparations in MT-4 cells, U937 cells, or donor CD4+ lymphoblasts was determined. Cells were inoculated with a twofold dilution series of virus, and eight replicates were cultured until the peak of virus production, as measured by antigen capture enzyme-linked immunosorbent assay (ELISA). An optical density three standard deviations above the mean for eight uninfected cultures was considered positive for infection. The CCID50 was defined as the reciprocal of the dilution at which 50% of the cultures were infected, using the Reed-Muench method (27). Additionally, plasma from late-stage HIV-infected patients was used directly as a virus source. Radioimmunoprecipitation analysis. A total of 6 x 106 H9 cells infected with HIV,11B or 6 x 106 uninfected H9 cells were cultured for 72 h in 8 ml of growth medium containing 500 ,uCi of [3H]glucosamine (Amersham; specific activity, 26 TABLE 2. Neutralization with MAb 1E3 of six HIV-1 and five HIV-2 isolates Virus strain

IC80

% Inhibition of infectiona

(ng/CCID5o)b

99 52 94 62 66 54

5.2 41.2 21.8 NDc ND ND

98 99 100 100 100

7.4 1.1 ND ND ND

HIV-1

HIVI1B

CDC451

SSI102

SSI121c

SS1122c

SS1146c HIV-2

SBL6669 LAV-2 SS1082 SS1131 SS1502

a Percent inhibition of infection relative to mock-treated controls at 25 ng/CCID50 for all isolates. b The 80% inhibitory concentration (IC80) was determined by interpolation from dilution series of MAb (cf. Fig. 1) for the established laboratory strains. c ND, not done.

20

-

0

-

-T,

n 0

1

3

9

nanogram

27

80

MAb per CCID50

FIG. 1. Infection of the lymphocytic cell-line MT-4 by HIV-1 isolate HIV111B was inhibited by MAbs specific for Tn. MAb 1E3 (solid bars) was an IgG2a immunoglobulin, and MAb TKH6 (open bars) was an IgM immunoglobulin. The HIV antigen concentration in culture supernatants 4 days postinfection was expressed relative to the antigen concentration in control cultures of mock-treated cells inoculated with mock-treated HIV. The results are the means of quadruplicate cultures SEM in a representative experiment. ±

Ci/mmol). Cells were washed in PBS and sonicated for 2 min in 1 ml of lysis buffer (1% Triton X-100, 10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 100 IU of aprotinin per ml, 0.2 mM phenylmethylsulfonyl fluoride), and 50 RI of lysate was incubated at 4°C overnight with 3 ,ug of MAb 1E3 against Tn, TKH2 and B72.3 against sialosyl-Tn (17, 22), positivecontrol MAb 110-4 against gpl20 (Genetic Systems, Seattle, Wash.) (16), and negative-control MAb against human glial fibrillary acidic protein (a-GFAP, Dakopatts, Copenhagen, Denmark). Then 100 ,ul of protein A-Sepharose was added, and after incubation for 3 h, pellets were washed five times in 10 mM Tris-NaCl-1% Triton X-100 (pH 7.6) and used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 8% homogeneous gels. After incubation in amplifying mixture (Amplify; Amersham) and drying, gels were placed on Hyperfilm MP (Amersham) films for 10 days. Inhibition assay. A total of 106 MT-4 cells in 500 pL. of growth medium were inoculated with 25 CCID50 of HIV for 2 h. With the HIV-2 isolates SBL6669 and LAV-2, an inoculum of 100 CCID50 was also tested. Prior to inoculation, either the cells or the viral inoculum was preincubated for 1 h with an MAb dilution series. MAb-preincubated cells were washed before inoculation. After inoculation, cells were washed and quadruplicates of 200,000 cells were cultured in growth medium without MAb in 24-well cell culture plates. For inhibition assays with the fresh HIV-2 isolates, CD4+ donor lymphoblasts were used as target cells. Inhibition of infection in U937 cells was assayed in a similar manner: 2 x 106 U937 cells were inoculated with 25 CCID50 of SSI002 and subsequently cultured in quadruplicate. Infection of U937 cultures was evaluated at day 10.

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percent infection relative to control

percent infection relative to control

140140

120 120-

1

100

100

80

60-

60-

40-

40 20-

20

0T 0

0.3

0.9

2.8 nanogram

8.3

25

MAb per CCIDso

FIG. 2. Infection of MT-4 cells by HIV-2 isolate SBL6669 (solid bars) and LAV-2 (open bars) was inhibited by MAb 1E3 specific for Tn. The HIV antigen concentration in culture supernatants 5 days postinfection was expressed relative to the antigen concentration in control cultures of mock-treated cells inoculated with mock-treated HIV. The results are the mean of quadruplicate cultures SEM in a representative experiment.

Dose-response titration was performed with all established strains (HIV,,,B, CDC451, SSIO02 SBL6669, and LAV2), whereas fresh isolates were tested with MAb 1E3 at a concentration of 25 ng/CCID50, and inhibition of infection was defined as the presence of less than 50% HIV antigen (mean of four replicates) in culture supernatants 4 to 6 days post infection. In a separate series of experiments, heparinized plasma (5 IU/ml) from two patients with AIDS (CDC stage IV) was used directly to infect phytohemagglutinin-stimulated cultures of donor CD4+ lymphocytes selected by "panning" (21): 0.5 ml of plasma was preincubated for 1 h with hybridoma supernatant containing 10 ,ug of MAb or with growth medium as a control and used to inoculate 2 x 106 CD4+ lymphoblasts in a total volume of 1.5 ml for 3 h. After being washed, the cells were cultured in quadruplicate for 2 weeks and evaluated for infection as described above. ELISA. The amount of HIV antigen in cell-free culture supernatants in 96-well immunoplates was routinely measured by using a double-antibody sandwich ELISA with human IgG primarily against p24 as both capture and detecting antibody as previously described (20). Each plate included a dilution series of a standard HIV antigen preparation, and optical densities at 490 nm were expressed relative to this standard preparation (arbitrary units). All in vitro infection experiments included a control in which untreated HIV was used. After 4 (MT-4 cells), 6 (donor lymphoblasts), or 10 (U937) days of culture, supernatants were diluted to give an optical density at 490 nm of approximately 2 from this control. All antibodies used for inhibition of infection were also tested for interference with the ELISA detection-,

0

10

20

40

80

160

nanogram MAb per CCID5o FIG. 3. Infection of the monocytoid cell line U937 by HIV-1 isolate SSI002 was inhibited by MAbs specific for Tn. MAbs were of the IgG2a (1E3 [solid bars]) or IgM (TKH6 [open bars]) type. Infection was measured by HIV antigen concentration in culture supernatants after 10 days and expressed as a percentage of a mock-treated control-culture. The results are the mean ± SEM of quadruplicate determinations.

and no such interference was found. For comparison, selected experiments were also evaluated by using a commercial p24 antigen capture ELISA kit (Abbott); the results were identical. Inhibition of attachment. A dilution series of MAb or PBS was incubated with 25 CCID50 of HIVIIIB for 1 h. These HIV preparations were then added to 1 x 106 MT-4 cells in 500 ,ll of growth medium and incubated for 1 h at 4°C to allow attachment of virus but prevent penetration (33). Cells were washed in cold medium, and PBS or MAb was added to cultures in which the HIV inoculum had been preincubated with MAb or PBS, respectively. After a 1-h incubation at 4°C, cells were washed, transferred to 24-well cell culture plates in quadruplicate, and evaluated for infection as described above. Syncytium assay. Chronically HIVIIIB-infected H9 cells (10,000 cells per well) were incubated in 50 ,ul of growth medium containing a serial dilution of MAb 1E3 in a 96-well cell culture plate. After 1 h, 100,000 uninfected CD4+ lymphocytes from a normal donor were added in 50 Rll of growth medium. After 18 h, the number of syncytia (giant cells with more than five nuclei and ballooning cytoplasm) in each well was determined by using an inverse microscope (magnification, x 100). In addition to PBS, the MAbs Leu3a (Becton-Dickinson, Mountain View, Calif.) and OKT8 (ATCC clone CRL 8014) were used as positive and negative

controls, respectively. Validity. All experiments were performed at least twice with identical results; unless otherwise indicated, results from one representative experiment are shown.

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TABLE 3. Inhibition of HIV infection by anti-Tn antibody was a result of antibody binding to virus, not to cells' % Infection after preincubation Ofb HIV Cells

Antibody

2 ng of 1E3/CCID50 20 ng of 1E3/CCID50 0.3 mM GalNAc-Ser 2 ng of 1E3/CCID50 + 0.3 mM GalNAc-Ser

54.5 0.1 103.1 100.8

± ± ± ±

10.0 1.2 13.6 10.6

90.1 ± 6.6 98.2 ± 11.1 96.4 ± 8.3 NDC

a Inhibition was abrogated by GalNAc-Ser (Tn antigen) from Biocarb. Either cells (MT-4) or virus (HIVI1B) was preincubated with 20 and 2 ng of MAb 1E3 per CCID50 or with PBS. Before inoculation with 25 CCID50 of HIVIJB, preincubated cells were washed thoroughly. b Infection at day 4 was expressed as HIV antigen concentration in culture supernatants relative to a mock-treated control culture. The results are the mean ± SEM of quadruplicate cultures. Similar results were obtained with a different preparation of GalNAc-Ser. c ND, not done.

percent infection relative to control 140 120

100

80 60

40

20

RESULTS

MAbs against the carbohydrate Tn epitope (GalNAc-Ser/ Thr) inhibited both HIV-1 and HIV-2 infection in vitro (Table 2). Both an IgG2a (1E3) and an IgM (TKH6) MAb showed a dose-dependent inhibition of infection as evaluated by virus antigen production when the virus was preincubated with MAb prior to inoculation of either lymphocytic (Fig. 1 syncytia per well 120 1

100

80

60

40

20

0

0.3

0.8

0.002

0.02

0.2

2

pg/mI FIG. 4. Dose response of anti-Tn MAb 1E3 on syncytium formation. Chronically HIVI1B-infected H9 cells were incubated in a serial dilution of MAb 1E3. After 1 h uninfected CD4+ lymphocytes from a normal donor were added, and 18 h later the number of syncytia in each well were counted by using an inverse microscope (magnification x 100). In addition to PBS, the MAbs Leu3a against CD4 (Becton-Dickinson) and OKT8 against CD8 (ATCC clone CRL 8014) were used as positive and negative controls. The results are the mean of duplicate determinations.

0

1.5

3

12

0.5

nanogram MAb per CCID5o FIG. 5. Effect of MAb 1E3 on HIV111B infection of MT-4 cells when the viral inoculum was preincubated with MAb before (solid bars) or after (open bars) being mixed with the cells. A dilution series of MAb or PBS was incubated with 25 CCID50 of HIV111B for 1 h. These HIV preparations were then added to 1 x 106 MT-4 cells in 500 p.l of growth medium and incubated for 1 h at 4°C to allow attachment of virus but prevent penetration. Cells were washed in cold medium, and PBS or MAb was added to cultures in which the HIV inoculum had been preincubated with MAb or PBS, respectively. After a 1-h incubation at 4°C, cells were washed and transferred to 24-well cell culture plates in quadruplicate. The results are the mean HIV antigen concentration in quadruplicate cultures + SEM at day 4 relative to a mock-treated control culture.

and 2) or monocytoid (Fig. 3) cells. For syncytium-forming isolates, anti-Tn MAbs also blocked syncytium formation in cultures inoculated with free virus, which had been preincubated with MAb, at the same concentrations of MAb which inhibited HIV antigen production (data not shown). Isotype matched carbohydrate-specific control MAbs TH1 (IgG2a) and TKH5 (IgM) had no effect on infection as evidenced by 107 and 121% HIV,,,B antigen production at as high as 1,000 ng/CCID50. Although inhibition of infection by using IgG MAb 1E3 was identical with lymphocytic (MT-4) and monocytoid (U937) cells, the dose response of IgM MAb TKH6 showed less efficient infectivity inhibition when monocytoid cells were used than when lymphocytic cells were used (Fig. 3). In MT-4 cultures, in which HIV-induced cytopathic effects are readily detected, inoculation with MAb-treated HIV produced live cell counts that were typically two to three times higher after 1 week than in corresponding cultures inoculated with untreated HIV (data not shown). Dose-response titrations of MAb 1E3 against established HIV-1 and HIV-2 strains showed similar 80% inhibitory concentrations in the range of 1 to 40 ng of MAb per CCID50 (Table 2). When MAb 1E3 was used at a fixed concentration of 25 ng/CCID50, three fresh HIV-1 and HIV-2 isolates were also inhibited (>50% inhibition compared with controls).

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VOL. 65, 1991

percent infection relative to control 120

100

8060

-/

40-

20 0 0

6

50

25

12

nanogram MAb per CCID5o FIG. 6. The anti-carbohydrate monoclonal antibody 1E3, specific for the Tn epitope, inhibited infection with HIVIB which had been passed in peripheral blood mononuclear cell cultures from histo-blood group A, B, or 0 donors. The HIV,,,B isolate was passed twice in PBMC from either blood type A, B, or 0 donors, and supernatants HIVI1B/lyA (solid bars), HIV11,B/lyB (open bars), and HIV11,B/lyO (hatched bars) from these cultures were used for the inhibition assay. The results are the mean of quadruplicate cultures + SEM.

Protection against infection was a result of MAb binding to virus rather than to cells, since preincubation of target cells with MAb and subsequent washing prior to inoculation had no effect on their susceptibility to infection (Table 3).

The anti-Tn MAbs were also effective in inhibiting the formation of syncytia between infected and uninfected cells. After preincubation of permissively HIV111B-infected H9 cells, formation of syncytia in a coculture of these cells and CD4+ lymphocytes from healthy donors was inhibited in a dose-dependent fashion (Fig. 4). The specificity of the MAb-mediated inhibition was examined by using synthetically produced, pure GalNAc-Ser (Tn hapten). When 2 ng of MAb 1E3, which produced 50% inhibition in itself, was incubated with 0.3 mM GalNAc-Ser prior to incubation with virus, no inhibition of infection occurred (Table 3). The mode of action of MAb 1E3 was examined by using inoculation at 4°C. This showed (Fig. 5) that MAb 1E3 was effective in blocking infection only when incubated with virus prior to inoculation. No inhibitory effect was found when the MAb was added after the virus had been allowed to adhere to the target cells at 4°C for 1 h. Because the expression of carbohydrate antigens on HIV has recently been shown to be related to the histo-blood type of cells used for virus propagation (3), the HIV,,,B strain was passed in lymphocytes from healthy donors with histo-blood type A, B, or 0. Virus preparations from these culture supernatants were equally susceptible to MAb-mediated neutralization (Fig. 6). To examine the theoretical possibility that HIV obtained the Tn structure as a result of in vitro isolation or passage, we used plasma from late-stage HIV-infected patients as the virus source. Plasma was obtained from five AIDS patients, but only two of these samples produced infection in CD4+ lymphoblasts and none infected MT-4 cells. When 0.5 ml of infectious plasma was preincubated with 10 pg of MAb 1E3, infection was completely abrogated. Finally, the viral component expressing 0-linked carbohydrate structures recognized by the neutralizing MAb 1E3 was examined. Lysates from HIVIIIB-infected and uninfected cells labeled with [3H]GlcN were precipitated with MAbs against Tn and sialosyl-Tn (Fig. 7). Although only a single band at 70 kDa was nonspecifically precipitated from both infected (Fig. 7A) and uninfected (Fig. 7B) cells, gpl20 was specifically precipitated by MAb 110-4 (Fig. 7A, lane 4) against a peptide epitope of gp120, MAb 1E3 (lane 1) against Tn, and MAbs TKH2 and B72.3 (lanes 2 and 3) against sialosyl-Tn. Additionally, specific bands at 140

B

A 1

2

3

6465

4

1

5

2

3

4

5

-12C00 -200

gp12O-

93

-

69

-

46

-

93

-

69

-

46

FIG. 7. Cell lysates of HIV111B-infected (A) or uninfected (B) H9 cells labeled with [3H]glucosamine were incubated with MAbs and precipitated with protein A-Sepharose. Lanes: 1, MAb 1E3; 2, MAb TKH2; 3, MAb B72.3; 4, positive control MAb 110-4; 5, negative control MAb a-GFAP (5). Precipitates were used for SDS-PAGE with 8% homogeneous gels and subsequent fluorography.

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and 160 kDa were visible when MAb 1E3 and TKH2 were used, indicating the presence of simple 0-linked carbohydrate structures not only on gp120 but also on the precursor gp160.

DISCUSSION

Other retroviruses are known to contain 0-linked glycans in their envelope glycoproteins (24). Some indications have previously been found that small amounts of sialylated 0-linked glycans exist on HIV gpl60/gpl20 (31) and that these structures are neutralization epitopes in vitro (12). In this study we have looked for the simplest possible 0-linked glycan, Tn (GalNAc-Ser/Thr), as this structure is very restrictively expressed in healthy humans (17, 30) and as Tn can induce both a humoral as well as a cellular immune response in mice (28). Both gp160 and gp120 contained the Tn structure, since MAb 1E3 against Tn precipitated gp120 and gp160 from HIVIIIB-infected and radiolabeled cell lysates. This fits well with the location to the cis-Golgi of initiation of 0-linked glycosylation (23) and proteolytic cleavage of gpl60 to gpl20 and gp4l in the cis/medial-Golgi (31). The Tn structure was found to be a broadly specific neutralization epitope, since IgG and IgM MAbs against Tn neutralized a number of HIV-1 and HIV-2 isolates and also blocked fusion between HIV-infected and uninfected cells. The neutralizing concentrations of anti-Tn IgG MAb 1E3 (80% inhibition at 1 to 40 ng/CCID50) were similar to the neutralizing concentrations of specific IgG MAbs against the V3 region (80% inhibition at 0.1 to 500 ng/CCID50) as reported previously (la). Although type-specific neutralizing V3 antibodies do not block attachment (26, 29), indications were found that the neutralizing effect of anti-Tn MAb occurred by specific binding of MAb to virus and that anti-Tn MAb probably interfered with infection at a step prior to penetration. Tn is a primitive 0-linked glycan expressed on cancer cells as a neoantigen (17, 30). Since glycosylation of viral glycoproteins utilizes cellular glycosyltransferases, the Tn epitope could theoretically be expected to be present on HIV produced in transformed cell lines. Thus the cell line H9 produced HIV111B, which was susceptible to neutralization with anti-Tn MAbs. However, after passage of this virus in lymphocyte cultures from normal donors, which has recently been demonstrated to adjust viral glycosylation to the glycosylation pattern of production cells (3), the passaged virus was fully susceptible to neutralization by anti-Tn MAb. Using plasma from AIDS patients, we furthermore found that virus as it is produced in vivo was also susceptible to neutralization by anti-Tn MAb. This excludes the possibility that the Tn structure was incorporated into the viral envelope as a result of in vitro passage of virus. This leads us to suggest that HIV infection in itself may induce expression of the carbohydrate neoantigen Tn. Since this antigen is a precursor to the sialosyl-Tn antigen, which is also a neutralization epitope, we propose that HIV infection may lead to either expression of abnormal glycosyltransferases or abnormal termination of glycosylation processing in the Golgi. Viral glycoproteins may thus obtain increased amounts of incompletely processed oligosaccharide chains, which will present themselves as carbohydrate neoantigens. Similar induction of neoglycosylation has previously been found for cytomegalovirus and HIV (1, 2), and murine leukemia virus has been shown to effect alterations

mediated by the ras oncogene in the glycosylation process in infected cells (5). Although anti-sialosyl-Tn MAbs have previously been found to neutralize infection of lymphocytes (12), one antisialosyl-Tn MAb enhanced infection of U937 cells with HIV-1 isolate SS1002 (13). Whether such enhancement in vitro is of real importance in vivo is not clear, but for vaccines it is at least worrisome. In this study we therefore also examined the effect on U937 infection with HIV SSI00, and although IgM anti-Tn was less effective than IgG anti-Tn in neutralizing infection, no evidence of enhancement was found. A low-titer immunoreactivity against Tn is found in most human sera (30). Since this study demonstrated effective in vitro neutralization when using antibodies specific for this carbohydrate antigen, boosting of the naturally occurring low-titer anti-Tn immunoreactivity may be of interest as infection prophylaxis. ACKNOWLEDGMENTS We thank Sen-Itiroh Hakomori for his support and Katja Bergholdt, Alice S. Nielsen, and Anne L. Jensen for excellent technical assistance. The study was financially supported by The Danish Medical Research Council, The University of Copenhagen, The Danish Insurance Association, and The Lundbeck Foundation.

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